HIrudin AA Sequences, CDNA Cloning, Genomic Organization

8/8/2019 HIrudin AA Sequences, CDNA Cloning, Genomic Organization

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2 %amino acid sequences were determined with microanalyticalmethods. On the basis of structural information, we appliedthe polymerase chain reaction (PCR) technique to isolate thecDNAs coding tor the hirudin-like polypeptides HMl andHM2. In addition, by cloning the genomic fragments of HMland H1VI2. we have elucidated for the first time the geneorganization of hirudin-like polypeptides. Fully active re-combinant HM2 was also produced in Esctierichia coti cellsfollowing transformation with a synthetic gene and using aperiplasmic expression system.

MATERIALS AND METHODSLeeches

Starved H. manillcnsis leeches were provided by Bio-pharm UK. Leech heads were dissected out from bodies,washed in ."> M NaCl. and quickly fro/en in liquid nitrogenprior to storage at -8()"'C.Isolation of antithrombin polypeptides

Crude extracts were prepared from the head part of theleeches essentially as described previously |14|. The purifi-cation process was monitored by assaying the antithrombinactivity.Lyophili/ed extracts were dissolved in 20 mM amm o-nium formate pH 3.0. and applied to a Mono S HR 5t5 col-umn (Pharmacia) equilibrated in 0.1 M ammonium formatepH VO. Elution was carried out increasing the pH to 4.5with a linear gradient in 60 min. Pooled active fractions werelyophili/ed. dissolved in 20 mM ammonium formate pH 7.0and loaded onto a Mono Q HR 515 column (Pharmacia). Stepelution w as carried out w ith 0.1 5. 0.4 and 0.8 M NaC l inamtTionium formate buffer.Bovine thrombin (Sigma) was further purified accordingto a described procedure \\5\ and immobili/ed to activatedSepharose CL 6B following manufacturer's instructions. Theactive pool from Mono Q chromatography was adjusted topH K.3 and applied to a small column (1.7 ml) of immobi-li/ed thrombin equilibrated in 50 mM Tris/HCl pH S.3. Thecolumn was washed with lO-ml aliquots of Tris buffer. Tris/3 M NaCl and Tris buffer.Bound m aterial w as then eluted with 25 mM 4-am ino-ben/am idine and desalted against 10 mM sodium phosphatepH 7.0 on a Smart system using a fast desalting PC 3.2/10column (Pharmacia). The last step of purification was per-lonned by reverse-phase high-performance liquid chroma-tography (RP-HPLC) using a C, Vydac column (4.6X

250 mm . 5 ^m ). Antithrombin polypeptide isoforms wereseparated w ith 20 mM sodium pho sphate pH 7.5 (eluent A)and 95'/r acetonitrile (eluent B) in a linear gradient from5'/' to 25'/f B in 30 min. Fractions co rresponding to purifiedisoforms were concentrated under vacuum.Structural analysis of antithrombin polypeptides

Purified antithrombin polypeptides (5-.5()ng) were re-duced with a 50-fold molar excess of dithiothreitol/6 M gua-nidine in Tris/HCl pH K.5 and .V-pyridylethylated by reactingwith 4-vinylpyridine |16|.Derivati/cd or intact polypeptides were digested withtrypsin (treated with tosylphenylalanine chloromethane) orwith Siaphxiococcus aureus VH protease (Sigma) |1 7| . Theresulting peptide mixtures were lyophili/ed and subjected to

peptide mapping by RP-HPLC on a column of Waters |iBondapak C,K (3.9 X 300 mm. 10

N-terminal .sequence analysisN-terminal sequence analysis of intact or .S-pyridylethylated antithrombin polypeptides and tragments generatethereof by en/ymatic cleavages was performed by automate

Edman degradation using a pulsed liquid-phase sequencemodel 477A with an on-line analy/er model 12()A (ApplieBiosystems) tor the detection of phenylthiohydantoin derivatives of amino acids (Pth-Xaa). Standard manutacturers' programs were used with minor moditications.

C-terminal sequence analysisC-terminal sequence analysis was performed atter timcourse digestion of intact antithrombin polypeptides [510|ag) with carboxypeptidase P (Boehringer Mannheim) aprevit)usly described |181.

Biochemical analyse.sAntithrombin activity was assayed by two //; virro testThe first one was based on the inhibition of thrombin-catly/ed conversion of fibrinogen to an insoluble clot and thresults were expressed in antithrombin units (ATU) 119|. Thother test measured the inhibition of thrombin-mediated rlease of /j-nitroanilide from the chromogenic substrate 2238 (Kabi-Vitrutn) and the results were expressed in thrombin inhibition units (TIU) |2()|. A working standard of thrombin solution was calibrated against a specimen of First Iternational Standard of human thrombin (National Institutor Biological Standards and Controls. South Mimms. UK)The amino acid compositions of polypeptide preparatiowere determined by analysis of the phenylthiocarbamoyl drivatives of amino acids (Ptc-Xaa) atter vapor-phase hydrolsis with 6 M HC l. Automated Ptc-Xaa a nalysis w as petbrmed on an Applied Biosystems amino acid derivati/model 420 A equipped with an on-line HPLC. Protein cocentration was determined with the Bradtbrd procedure |2tusing bovine serum albumin as a standard.

Isolation and characterization of RNA and DNATotal cellular RNA was prepared trom leech heads essetially as described by Harvey et al. |13|.DNA was extracted from tro/en //. nidnittensis leechaccording to the Applied Biosystems protocol tor DNA/RNextractor.For Southern an alysis 10 ng genomic D NA w as used teach digestion tbilowing incubation conditions recommended by the manufacturers. Restriction fragments weseparated on a 0.8% agarose gel and transterred to a nitrocelulose tilter (Millipore) essentially as described by Sambrooet al. |22|. Standard pre-hybridi/ation and hybridi/ation coditions were used |22|; the ' 'P-labeled probe was prepareby the random prime method using the multiprime DNA lbelling kit (Amersham). The filter was washed to a finstringency of NaCl/Cit (NaCI/Cit is 0.15 M N aCl. 0.015 trisodium citrate. pH 7.0) 0. 1% SD S, at 65"C tbr 30 m

and autoradiographcd at -8()"C' using Hypcrliliii M(Amersham).


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2cDNA .synthe.sis

Thc reverse transcription reaction was carried out in a40-^ 1 volume as follow s: 10 (ig total RNA from leech headswas tnixed with 1 (ig oligo(dT) pritner. 8 )il 5 niM dNTP mixand 8 (tl reverse transcriptase buffer (250 niM Tris/HC l pH8.3. 300 mM KCl. .50 mM Mg CI,. 5 mM dithiothreitol).heated to 65 C for 2 min and qu ick chilled on ice. 10 URNasin (Protnega) and 20 U avian myeloblastosis virus re-verse transcriptase (Boehringer Mannheim) were added, andthe tube was incubated at 42C for 2 h. The reaction mixturewas phcnol/chloroform-cxtt acted, isopropanol-precipitatedand lesuspended in 60 (.tl sterile distilled water.Oligonucleotide.s

Oligonucleotides primers were synthesized on an A ppliedBiosystctiis model 38()B DNA synthesizer. Primers utilizedfor amplification of cDNA sequences were the following:dT17 adaptor primer. 5' GACTCGAGTCGACATCGATTT-TTTT TTTT TTTT TT3' : adaptor pr imer. 5 ' GACTCGA GTC-GAC ATC G X: (irimer 3 - 8 . 5' ATCGAAG CTTA(TC)AC-(CAT)GA(TC)TG(TC)ACNGA 3' ; pr imer 5 6 -5 2 . 5 ' CTAA-QQAIC C TTC (TC )TC (GA)AA(GA)TC NC C 3 ' ; p rimer 3 2 -37 5 ' ATC GGAATTC AGTTC TGGAAATC AGTGC GT 3 ' :pr imer 5 'RT. 5 ' CTAAGAATTCTTCGCAACTTATATGC-GTT 3 ' : p rimer 64 -6 0 . 5 ' ATC GGAATTC TTAATTC AAT-ATATCTTCAT 3'. Primer for DNA amplification wasprimer signal peptide 1 - 7 . 5 ' AGTC GAC AAATGTTC TC T-CT CA AG TTG TTC 3'. The restriction sites added at the .5'end of each primer to facilitate cloning and sequencing ofthe amplification product are underlined. N denotes a mixtureof all four nucleotides.PCR amplifications

To obtain the complete sequence of HMl and HM2cDNAs. three rounds of PCR amplification were perfonned.Eirst round

5 (il reverse-transcribed RNA was amplified in a mixturecon tainin g 10 (.tl lOX PCR bu ffer (C etus/P erkin Elm er). 2 (tl0.1 M M gCl.. Un tI dN TP tiiix (1.25 tiiM each dN TP) and500 pmol degenerated primers 3 - 8 and 56 -5 2 . Prior to add-ing 2.5 U Tciq polymerase (Cctus/Perkin-Elmer) the leactionmixture was heated at 95 C for 5 min and then allowed tocool at 70"C. Amplification was achieved with a cycle ot 1-min denaUiration at 94"C. 2-min annealing at 6()C and 2.5-min polymera.se extension at 72 X" for a total of 30 cycles,with a final 7-min 72" C extension step, using a Cetu.s/Perkin-Elmcr DNA thetmal eyelet.

Third roundTo clone the 5' end of HMl and HM2 cDNAs. we alutilized the RACE protocol. Reverse transcription of heRNA was performed using the 5'RT primer, as previousdescribed. The reaction mixture was then isopropanol-precitated and the first-strand cDNA products were polyadenlated at their 3' ends using terminal deoxynucleotidyltranferase (BRL) | 2 3 |. 10 (il ofth e polyadenylated products wamplified using 10 pmol dT17 adaptor primer. 25 pmol adator primer and 25 ptnol second-gene-specific primer ( 660) up stream to the first used for transcrip tion: 10 (il of tamplification product was then reamplified in the same coditions.

PCR amplification on DNA2 (ig leech DNA was amplified in the presence 100 pmol 1 - 7 signal peptide pritner and the 6 4 - 6 0 primas described (24]. Each cycle of PCR included DNA denatuation at 94C for 30 s followed by prime r annealing for 15and polymerase extension at 72C for 30 s. The annealintemperature was gradually increased: for the first five cycle

45C. 50C for other five cycles and then 55C for 25 cycles.Analysis of PCR products

Amplified products were analyzed on \.59r agarose gephenol-purified and ethanol-precipitated. After cleavage arestriction sites present in each primer, they were subcloneinto pUC vector, using standard procedures |22|. Sequencewere obtained on both strands using the sequenase ki(United States Biochemicals).For Sou thern blot analy sis. 15 (.il amp lification pro duc twas subjected to electrophoresis on a 1.5% agarose gel andsubsequently transfeired onto a Hybond-N ' tnembrane (Am

ersham) as described in the manufacturers' protiKoI. The '-Plabeled probe was prepared as described before. After hybridization, conducted using standard co nditions |2 21 . thmembrane was washed at increasing stringency up to0.05 X NaCl/Cit. 0.1 9r SDS at 65 "C .Recombinant DNA techniques

All DNA manipulations were carried out essentially asdescribed by Satnbrook et al. |22|. Oligonucleotides weresynthesized using an automated DNA synthesizer tnodel38()B (Applied Biosystems) and purified by polyacrylamidegel electrophoresis. Oligonucleotides were subcloned in aM13 vector and their sequences were verified using theseqtienase kit (United States Biochetnicals).Second round

Amplification of cDNA 3' ends was achieved followitigthe method of rapid amplification of cDNA ends (RACE)| 23 | . IOO pmol gene-specific pritner (3 2 -3 7 ) w as used to-gether w ith 100 pmol dT 17 adap tor primer to amplify 5 ^i \first-strand cDNA. as previously described. 5 (.il (if this am-plified product was subjected to a second amplification inthe same conditions but using a more stringent cycle: 1.5-min denaturation at 94"C followed by 2.5-min annealing ex-tension at 72"C. Alter 30 cycles, the reaction tnixture wasextended for 7 min at 72 X\

Expres.sion and purification of recombinant HM2A 4-1 fennentation of recotnbinant E. coli strain B werecarried out in a tettacycline-co ntaining mediutn. for 8 h at37C. Bacterial cells were harvested by low-speed centrifu-gation and stored at -2()''C utitil used. The periplastniccontent was released by stirring the whole cell paste in Ibr-mate buffer pH 3. Raw ex tracts w ere first purified on aSepharose-S column at pH 3.1 with an increasing salt gradi-ent (0 - 1 M N aCl). After adjustment at pH 7. the recombi-nant HM2-containing fractions were loaded on a Sepharose

6B column chelating Cu ' ions (Phartnacia). previously


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29 80 . 3 -

s0 . 2 -

I "'

-

1

a

2

t

i li'

i l l , ,

Table 1. Amino acid composition of H. manillensis antithrombins. Cys was determined as .S'-pyridylethylated derivative; n.d.. nodetermined.

t 0 t 5 2 0 2 5time (min)

Fig. 1. RP-HPL C purification of H. manillensis antithrombinpolypeptides. Following thrombin-Sepharose chromatography, theactive pool was purified on a C4 Vydac column eluted with a sodiumphosphate/acetonitrile system at neutral pH as described in Materialsand Methixls. The three indicated peaks have been respectively des-ignated HMl. HM2 and HM3.

equilibrated in 50 mM sodium phosphate pH 7 con taining0.5 M N aCl. The metal-chelating column was stepwisceluted with phosphate buffer of decreasing pH. HM2 waseluted in the pH 4 phosphate step. Buffer exchange in ammo-nium bicarbonate was carried out on a Sephadex G-25 col-umn and solid HM2 was obtained by lyophilization of vola-tile bicarbonate buffer. The purity of the final HM2 prepara-tion was assayed by RP-HPLC and N-terminal sequenceanalysis.

RESULTSPurification and amino acid sequencingof antithrombin polypeptides from H. manillensis

Antitbrombin polypeptides were isolated from crude ex-tracts obtained from tbe head parts of H. m anillensis leeches.Atler acetone/acid extraction of the crude material, two stepsot ion-exchange chromatography were carried out. First thecrude extract was purified by cation exch ange on a Mon o Scolumn eluted by increasing the pH from 3 to 4.5. The activep(H)l was then chromatographed on a Mono Q column usinga stepwise gradient of NaCl. The active purified material wassubjected to affinity chromatography on thrombin-Sepharosein (irder to isolate only ttiose polypeptides which specificallybind to thrombin. The antithrombin polypeptides were fur-ther tractionated by RP-HPLC on a C, Vydac column elutedat neutral pH. This step was essential to separate the isoformsfrom each other. Thre e main active peaks (labeled H M l.HM2 and HM3) were obtained by RP-HPLC of the materialpurified by affinity chromatography (Fig. 1).

HMl and HM2 were then obtained in homogeneous formatter re-chromatography and subjected to structural analyses.When direct N-terminal sequencing of 1-nmol samples ofalkylated HMl and HM2 was performed, the tirst 42 and45 amino acid residues, respectively, were unambiguouslyidentified, with the exception of cycle 43 of HM2 analysiswhich gave no identifiable Pth-Xaa.Time-course hydrolyses of the native proteins with car-boxypeptida.se P resulted in the release of the same residuesfrom the C-terminus. suggesting the following C-terminal se-

Aminoacid

A sxGlxSerGlyHisArgTh rAlaPr oTy rVa lM etC y sli eLe uPheLysTr p

HM-1hydro-lysis sequence

residues/molecule7 .09.63.49 .20 .60.122.70.213.02.12. 3L 35. 61.92 .91.02. 8n.d.

10941110403231623140

HM-2hydro-lysis

8. 011.26 .6K.20 .80.20.32.5L73 .30 .35.5L 3.00.92.5n.d.

sequence

9117.S10403.14I)6'2310

quence: -(Glu. Asp)-Ile-Leu-Asn-COOH. Amino acid analysis showed a few significant difterences between HMl anHM 2 composition (Table 1). especially the presen ce of onmethionine residue in HMl.These preliminary structural analyses revealed abou70% of the whole amino acid sequence of bolb isoforms (aexpected for a birudin variant of about 60 amino acids) anindicated that the most marked differences concerned thcentral part of the two polypeptides between residues 20 40 . Determination of tbe complete amino acid sequence waachieved by peptide mapping analysis of HM l and HM2 performed after reduction and alkylation of cysteine residuewitb 4-vinyl-pyridine. Two different enzytnatic treatmentcleavage with trypsin and .V. aureus V8 protease, were employed in order to generate the proper overlapping fragmentFig. 2 (in Supp lemen t) show s the RP-H PLC profiles of thtryptic maps obtained tor the two polypeptides. The peptideisolated in sufficient yields from both tryptic and V8 proteasdigestion were subjected to N-terminal sequence analysis anthe complete amino acid sequences of HMl and HM2 werfinally determined (Fig. 3). Several features can be illustrateby the comparison of the five sequences shown in Fig. 3. (aThe sequences of HMl and HM2 differ in 10 atnino aciresidues all occurring in the central part of the polypeptide(between positions 19 and 39) and theretbrc share a similarity of about 84%; their sitnilarity with hirudin variant (HVl) is 64% and 70%. respectively, (b) The six cysteinresidues are found at the same position for all of the fivvariants, (c) There are no potential sites tor sulfation in thC-terminal part of HMl and HM2. as they lack the tyrosinresidue present in HVl at position 63.The threonine residue at position 43 of HM2 could nobe directly identified, since it gave no identifiable derivativfor the corresponding cycle during N-terminal sequencinand its presence was interred by amino acid analysis of tryp


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2

HVlHV2HV3HMlHM2

VnlH elieValVal

ValThrThrSerSpr

Ty rTy rTyrTyrTyr

Th rTh rThrThrTh r

AspAspAspAs pAsp

CysCysCy sCysCys

Th rThrThrThrThr

GluGiuGluGluGlu

SorSerSe rSerSe r

GlyGlyGlyGlyGly

Gi nGinClnGinGi n

As nAsnAsnAsnAsn

Va lVa lVa lLe uVa l

CysCysCysCysCys

Gl yGl yGl yGl yGly

GinLysLysGlyGlu

GlyGlyGlyGlyGly

AsnAsnAsnLysLys

LysLy sLy sHi sAs n

CysCysCysCy sCys

Th rTh rTh rAspHi s

Gl yGlyGlyGl yGl y

GluGluGluGluGlu

GlyGlyGlyGlyGly

ThrThrThrThrTh i

ProProProProPro

LeuLeuLeuTyrTyr

H e Leu Gly Ser AspH e Leu Gly Ser AsnH e Leu Gly Ser GinGlu Met Asp Gly SerGin Leu Ser Ser Ser

CysCysCysCysCys

Le uLeuLeuI.euLeu

CysCysCysCysCys

GluGluGluValVal

GlyGlyGlyGlyGly

SerSerSerGlySer

AsnAsnAsnAsnAsn

GlyGlyGlyGlyGly

GluLysLys

LysGlyAsp

AsnAsnAsnAsnAsn

GinGi nGi nLy sGin

CysCysCy sCysCy s

Va lVa lVa lVa lVa l

LysAsnLys_.y sLys

ProProProProPro

GinGluGl nLy sLy s

SerSe rSerSe rSe r

Hi sHisH I SGi nGi n

As nAs nAs nTh rTh r

AspAs nGinGl uGl u

Gl yGlyGlyGlyGl y

AspAspAspAspAsp

PhePhePhePhePh e

GluGluGluGluGlu

GluGluProGl uGlu

H eH eH eH eH e

ProP I oProProPro

Glu Glu . Tyr Lru GinGlu Glu . Tyr Leu GinGlu Asp Ala Tyr Asp GluAs p Gl u A sp lie l.f'u AsnAn p Gl u A::p 1 l Leu Asn

Fig.3. A comparison of amino acid sequence of some hirudin variants with HMl and HM2. Conserved residues are boxed. Spacare introduced tor maximal alignment.

tic peptide 21-Al. RP-HPLC purification of tryptic peptidesfrom HMl gave two peaks corresponding to the same resi-dues 37-47. One of them was completely sequenced andwas shown to have a thrconine residue at position 43. Forthe second peak thc presence of thrconine could not be dem-onstrated by direct N-terminal sequence analysis of the pep-tide but it was deduced by amino acid analysis as in HM2(see Fig. 2 and Table 2 in Supplement). These results indi-rectly suggest the presence of a post-translational modifica-tion of Thr43.Each of the three peaks of protein obtained from affinitychromatography and separated by RP-HPLC was found to beendowed with antithrombin activity. When assayed with thefibrin clot test, thc antithrombin activity of homogeneouspurified HM2 (11 (MX) ATU/mg) was found to be approxi-mately the same as rHVl (11 .^(X) ATU/mg).Amplirication of RNA saniple.s

Thc existence of highly conserved regions in several hi-rudin variants and in HM l and H M2 (Fig. 3) was the basisfor the generation of specific primers for PCR amplificationon H. manillensis RNA.The whole PCR strategy used to isolate HMl and HM2cDNAs is outlined in Fig. A. For the first round of amplifica-tion, sense and antisense primers were designed to includeall possible nucleotide combinatiiins that could encode theconserved regions spanning amino acids 3 - 8 and 5b-52.The PCR amplification performed on reverse-transcribedleech head RNA gave a series of bands, where the mostabundant corresponded to the predicted molecular mass. Thisband was isolated and subcloned into the plIC vector. Se-quencing of purified clones yielded the expected HMl andHM2 cDNA portions.To obtain 3'-end cDNA sequences we applied thc RACFprotocol |23|. This method allows amplification of regionsof unknown sequence between a specific point in thc tran-script and the ^ or 3' end. A cDNA-specific primer, spanning

residues 32 37 . was designed on the basis ofthe previoudetermined HM2 sequ ence (Fig. 4). This was used togethewith the dT17 adaptor primer to amplify the reverse-transcribed RNA. A small amount of the PCR mixture was reamplified with the same oligonucleotides using more stringent conditions. Two major bands were selected and. aftesequencing, the 3' end of HM2 cDNA was determined.In order to clone the 5' end of HM1 and HM2 cDNAwe again followed the RACE protocol. Reverse transcriptioof leech RNA was performed using a gene-specific prime(.i'RT) designed trom the previously determined C-terminanucleotide sequence (see Fig. 4). The first-strand reactioproducts were polyadenylated at their 3' ends. Finally. PCRamplification was accomplished by adding another gene-specific primer (64-60) upstream to the 5'RT. Howeverfollowing electrophoresis on agarose gel. only a broad smeacould be visualized and even a second re-ampliflcation stecarried out in the same conditions did not yield any discretband. At this point, thc PCR mixture was used to perform Southern blot analysis using as probe the HM2 nucleotidsequence obtained during the tlrst round of amplificationTwo dis crete band s of ap proxim ately 3CK) and 360 bp w erfinally detected (Fig. 5). These were purified, cloned and sequenced leading us to the elucidation of the full-lengthcDN As for the two va riants (Fig. 6). The am ino acid sequences of HMl and HM2. as deduced from the cDNAs. areidentical to the ones obtained by direct chemical sequencing(results not illustrated).

Amplification of DNA samples and Southern analysisLittle is known of the genomic organization of hirudin orhirudin-like genes. The availability of cDNA sequences gaveus the opportunity to undertake the isolation of genom ic frag-ments from H. tiumillciisis.Accordingly, a new primer corresponding to the first

seven amino acids ofthe signal peptide and which could thusrecognize both HMl and HM2 was designed and chemically


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HM2 VSTTDCTESCQNYCLCVCSNVCCECKNCQLSSSCNQCVHCECTPKPKSQTEGDFEEIPDEDILN

I St round3- B

2na round 3 2 -3 7 OHgo dT

3ra round . .ollgoOT 64 60

cDNAs. A rcprcsc-niaiion of HM2 mRNA wilh the correspo

B1 2 3

- 1353- 1078- 872- 603

31 0278234194

-*- 360 bp- - 300 bp

(A )\5[i\ amplifica-es 1 and 2) and the HaelW digest of ft>Xil4 DN Ai) as molecular ma.ss standard. (B) Autoradiograph of the sameybridizin g band s of about 36() and 3(K) bp present in lanes 1

64

horesis where a single band of ap proxitnatcly 7(X) bp

in Fig. 6. both gene fragments contain the entire

All splice junc tions follow the 5'GT /3'AG splice ruleIntrons, as in many species including plants and Dm-

mentioning that the nucleotide sequences of the four eof the cloned genes were a confirmation of thc previodetermined cDNA sequences, thus eliminating any doubpossible nucleotide misincorporations introduced by thepolymerase.In order to confirm the presence of introns in these gwe performed a .Southern analysis on total DNA extrfrom H. manillensi.s head. The genomic DNA preparwas digested with EcoRX and Ham\\\ whose cutting were not found in thc PCR-amplified genomic fragmentprobe, we used thc lull-length cDNA for HMl. As showFig. 7, even a double digestion with EcoRX and Bani\\not yield any h ybridization signal at sizes below 180thus confirming that EcoRX and BamH\ restriction sitepresent in thc HMl or HM2 genes, are indeed external tgenomic fragments obtained by PCR amplification. The molecular mass of thc hybridization signals confirmeaddition, thc presence of intervening sequences in thc and HM2 genes.

Expression and prrtduction of H1VI2Fifficient production of recombinant HM2 was achusing periplasmic expression in /;. coli. according to a egy previously described [27].Six oligonucleotides, 83-HX) bases long, were prcphy chemical synthesis and ligated to construct a syngene for periplasmic expression of HM2. Thc nucleotidquence was designed on the basis ot preferential codon

in E. coli |28|. As shown in Fig. 8 (in .Supplement), thcthetic gene consists of thc oinpA ribosomc-hinding sitelowed by the ompA leader peptide and HM2 codinquences. Thc HM2 gene was then inserted into an expreplasmid under the control of thc Tip promoter and carthc tetracycline resistance marker./:. coli strain B, harboring thc HM2 expression plawas selected lor thc production of thc protein. The pritranslation product of recombinant bacteria consisted OmpA-lcadcr-pcptide-HM2 fusion protein, which wasported to the periplasmic compartment and correctlycessed, as demonstrated by N- and C-tcrminal anaStarting from I 1 bacterial culture, approxim ately 7

HM2 were obtained in thc raw periplasmic extract. A spurification protocol with two chromatographic steps gfinal homogeneous preparation of HM2 with an overall


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n F S L K L F U U F L B U C I C U S g f i URTCTTCTCTCTCflnCTICTTCCITCTCnCCTCCCTCTTTCCfiTCTCCCTCTCTCfl f iCCnGgtoogoooooettaa l t lg lc tUgtoogoa

S V T 0ojgaaagtgt tgat togoatgtcatgt t to t l t t tg t t tg t t t tggccaatcaaatgocct t t t t t t t t t tooat tcogTGf lCCTRCf lCTCn

C T E S G O H V C L C UTTGTRCGG flnTCnCCCCRGnnTTflTTCTCTRTGCG TGgtacgttcgoaatttoctattttttatgctttoaattccccaocoaatgctgtcgI - t -

C G N L C G G G K N C E n O G S G N K C U O G EootcttlogGGRGGTRRTCTCTGCGGTGGRGGCRRRCRTTGTGRRRTGGRCGGTTCTGCftRRTRnOTGCGTCGRTGGGGgtttgltttggca

- S - U - - E - - N - O L S S - - - O - - H - -

otttotgacaoaaattttgotatatttatataoaacaccotagttactcaoagaotgtctgactcooggaoootttattggQgtottttgtoo cc cget c 0 c

aaccccttttgtttoccgccooaattgotootottgcccgogttocatottoaottgaoogotootggcgoootottottgotcottaotat.. Q O . . . . . . . . .

C T P K P K S O T E G D F E E I P D E D I L NcotcottttoottgcogRflGGTRCTCCGRRGCCTRRGRGCCRGRCTGRRGGCGRTTTCGRRGRflBTCCCRGflTCRRGflTRTRTTGRRTIfia

Fig.6. Nucleotide and deduced amino acid sequence of HMl and HM2 cDNAs and genomic fragments. Exons are shown in cletters; introns are shown in lowercase letters. Ihe deduced amino acids, eticoded by exons. aie shown above the first ba.se ot each ccstarting with the first ATCJ triplet. Matching nucleotide and atnino acid sequences between the two DNAs are denoted as a dottedSpaces are introduced to maximize alignment.

1 2 3 bp

-6800-4500-4000-3200

- 2 1 0 0- 1800

7. Southe rn blot analysis of // . manillensis DNA. Genomiediuesteil with EcoRl (lane 1) . W(W/1II (latie 2) and EcoRV

(lane 3) . respectively. T h e blot w as hybridized with the ' P-H M l cDNA. .Sizes of fragments, in bp . were detertiiined by

to the 1-kb molecular mass standard and are indicated ate right.

In the purification of HM2 we took advantage of the

ected . HM2 does not bind to ininiob ili/ed Z n " ions, btttCii ' clielating tesin and ean be eluted in

iiatography followed by a C u' -chelating column .The complete amino acid sequence of recotnbinant HM2

ing, peptide mapping and time-course hydrolysis with boxypeptidase. using the methods described above fornatural molecules.Besides this, eleetrospray mass spectrometry analy.sepurified HM2 gave an experitnental molecular mass6797.25 0 .6 3 Da against the value of 6797.23 Da calated for the molecule with the six eysteines all involvedisulfide bridges (data not shown).The final lyophilized pteparation i>f HM2 displayed biochemical activity when its potency as a throtnbin inhibwas assayed. Antithtotnbin activity of reeonibinant HM2 5850 Tlil/mg when tneasured by the S-2238 chromogtest and 11 8(K) ATU/tng. when tneasured with fibrinogesubstrate. According to these values, thrombin inhibitionHM2 is fully comparable to the activity of other recotnbinhirudin variants [30).

DISCUSSIONMost of the polypeptides belonging to the family of hdins and characterized to date ha\e been isolated frotn European medicinal leech //. inetlicinalis |9. 10]. This lehas been employed in both tnedical practice and pharmactical research for many years. Among the possible phamialogically active substances produced by leeches, hirudin its analogs have generated a widespread interest in both dustrial as well as academic environments due to their stroanticoagulant activity. Indeed, hirudin is the most active a

specific inhibitor of matntnalian thrombin so far chartetizetl.However, simply on a theoretical basis. H. tiiedicina


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M)2ors acting against mammalian thrombin. H. medicinatis, intact, is primarily an amphibian leech [11] and thus one mayspeculate that the pool of hirudins produced by such a leechis adapted primarily to neutralize amphibian thrombin.Accordingly we focused our attention on a different leechspecies. H. manittensis. reported to be primarily a parasite ofmammals |tt| . In this paper we report the purification, thestructural characterization and the molecular cloning of twonovel hirudin-like polypeptides from the Asian leech H. ma-nillensis. We also describe a method to produce a fully activeand correctly processed form of recombinant HM2 in highyield by expressing in E. coti an OmpA-leader-peptide HM2 fusion.

The primary structure of these new polypeptides indi-cates that they share the same general characteristics of thepreviously k nown h irudin variants |10 1. Indeed , an overall707f similarity with HVl and the presence ofthe six cysteineresidues in highly conserved positions suggest the presenceof three disulfide bridges equivalent to those found for HVl.It is. however, interesting to note that most of the vari-ability concerns amino acid portions which are not directlyinvolved in the interaction with thrombin. as shown by theresolution of tridjmensional structure of hirudin-thrombincrystals [7|. All of the hirudin variants extracted from H.medicinatis and characterized to date contain in the C-termi-nal portion of the molecule a site of sulfation. that is a tyro-sine residue, for example Tyr63 of HVl. The lack of thetyrosine residue in the C-terminal portion of the new hirudinvariants from H. manillen.sis is an interesting finding, indicat-ing that such post-translational modification might haveevolved differently atnong ditferent sub-families of leeches.The fact that we were not able to clone from H. manillensi.sany hirudin isoform carrying tyrosine residues in the COOHdomain could also imply possible evolutionary ditferencesbetween this leech species and H. medieinati.s.

While this paper was in preparation. Steiner et al. l.' l)reported the characterization, from a leech purported to beH. manillen.si.K, of two other hirudin-like polypeptides whichshare low similarity with HMl and HM2. One of the de-scribed sequences also carries a potential sulfation site. Arelevant aspect of the hirudin variants described by Steineret al. was the presence of a O-glycosidic chain on a threonineresidue. Combined results from peptide mapping, amino acidanalysis and sequencing indicate that the corresponding thre-onine residue at position 43 of extracted HMl and HM2 islikely to be a putative glycosylation site. Taken together,these data support the hypothesis that hirudin-like polypep-tides from H. manillen.si.s are characteristically modified withan O-glycosidic chain on threonine. Besides, a family of iso-form variants seems also to be present in H. manitlensi.s. asis well documented for H. medicinalis.

To isolate cDNA and genomic clones from the leech H.manitten.sis we used the well-established PCR technologyusing primers based on the amino acid sequence. In this re-spect, it is surprising to note that the gene in H. medicinati.scoding for the most characterized hirudin isoform, HVl. hasnever been isolated, while only a partial clone coding for theisoform HV2 has been rept)rted by Harvey et al. | t 3 | .A quite important feature of our technical approach is thecapability of isolating PCR-amplified clones from total RNApreparations extracted from a very limited number of leeches.In some cases the head of just one specimen was sufficientto yield a quantity of messenger RNA from which we couldamplify and clone the desired cDNA. This is an extremelyimportant aspect since it may not be excluded that a certain

degree of heterogeneity at the genetic level could be presein a given set of leeches. Indeed, a possible leech heterogeneity could explain why we were not able to isolate anclone coding for the hirudin analogs described by Steiner al . 1311.The cDNA clones coding for the hirudin isotorms HMand HM2 indicate the expression of pre-hirudins of 84 aminacids where the first 20 residues constitute the signal peptirequired for extracellular secretion. Interestingly, the HMand HM2 signal peptides share a 100% similarity both at thamino acid and nucleotide level. In addition, from the HVcDNA sequence previously reported |13|, it is worth notinthat the partial signal peptide is also similar to the corrsponding sequence in HMl and HM2. These data suggethat the secretion signals responsible for the intracellular anextracellular transport of the hirudin isotorms may be highconserved from one leech species to another. Moreover, susimilarity may offer an extremely valuable technical opportnity to PCR-clone additional isoforms of hirudin frommanitten.si.s as well as from other leeches by simply usas 'upstream primers' oligonucleotides based on the signpeptide sequence.The amino acid divergence between HMl and HM2 limited to the central 'core' region of the hirudin isotormFrom previous structural studies on the interaction of hirudwith thrombin, it appears that this region is mainly responsble for the structural conformation of the molecule and it not directly involved in thrombin binding | 8 |. The genomorganization of HMl and HM2 indicates that the observamino acid diversity between the two isoforms is coded byprecise exon. Interestingly, introns are not ramdomly placbut they correlate with functional elements of the encodprotein.All data reported in this paper demonstrate that these twgenes are very closely related to each other and support thypothesis that HMl and HM2 may have evolved by geduplication. It will be interesting to elucidate the genomorganization of other hirudin isoforms, even trom othleeches, to raise considerations on possible evolutionapathways or alternative expression mechanisms.

SupplementTable 2. Amino acid sequence of tryptic peptides of HMl aH1V12. Peptid es genera ted by trypsin dige stion were resolv ed hy RHPLC (as shown In Fig. 2) and subjected to sequence analysis. Threspective initial yields are reported; X = residue not determined amino acid sequ encing ; * = fragment containing a putative gtycsylated threonine residue

HMt

HM 2

Fragment

t - 1 .t4 -262 7 - 3 63 7 - 4 73 7 - 4 7 ( * )4 8 - 6 4t - 1 31 4 - 2 62 7 - 4 74 8 - 6 4

Sequence

VSYTDCTESGQNYCLCVGGNLCGGGKHCEMDGSGNKCVDGEGTPKPKCVDGEGXPKPKS Q T E G D F E E IP D E D IL NVSYTDCTESGQNYCLCVGSNVCGEGKNCQLSSSGNQCVHGEGXPKPKS Q T E G D F E E IP D E D IL N

Yieldpmol.'i654(K)32 036 0t63t6 8'M 65 47 t222 0


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0.15 r

10 20Time (min I

eo.Q

0.50

0.25

0. 0

B27-47

iu

13

14-26-

48-64'

1 1

1 0 - w

10 20Time (mint

Fig. 2. C4-HPLC peptide m apping of trypsin digested HMl (A)and HM2 (B). Samples of .V-pyridylethylalcd HMI (6 ng) and HM2(l()|ig) were digested with trypsin for 2 h at MC and generatedfragtnents were purified by RP-HPLC on a C4 Vydac columti asdescribed in Materials and Methods. The asterisk indicates the pep-tide containing a putative glycosylated threonine residue. The aminoacid sequences determined for each ofthe labeled peaks are reportedin Table 2 (in .Supplement).

We would like to thank Prof. Piero Pucci (University of Naplesfor electrospray-mass spectrometry analysis of rHM2 and Jan Malyszko for oligonucleotide synthesis.

REFERENCES1. Haycraft. J. B. (1884) On the action of a secretion obtainedfrom the medicinal leech on the coagulation of the bkxxiProe. R. Soc. Umd. 36. 4 7 8 - 4 8 7 .2. Markwardt. F. (19.'i5) Untersuchungen uber Hirudin. Naturwis-senselmften 42. 537-. ' i38.3. Markwardt. F. (19.'i7) Die l.solierung und chemische Charakteri-sierung des Hirudins. Hoppe-Sevter Z Physiot. Chem. M)S147- l . ' i6 .4. Dodt. J.. Seemuller. U.. Maschler. R. & Fritz. H. (1985) Thecotnplete covalent structure of hirudin. Localization ot thedisulfide bonds. Biol. Chem. Hoppe-Seyter 366. 3 7 9 - 3 8 5 .5. Wallace. A.. Dennis. S.. Hofsteenge. J. & Stone S. R. (1989)Contribution of the N-terminal regions of hirudin to its interaction with thrombin. Biochemistry- 2S . 10079-10084 .6. Naski. M. C. Fenton. J. W.. Maraganore. J. R.. Olson. S. T. &Shafer. J. A. (1990) The COOH-terminal domain of hirudin.An exosite-derived fibrinogen. / Biol. Chem. 265. 1 3 4 8 4 -13489.7. Rydel. T. J.. Ravichandran. K. G.. Tulinsky. A.. Bode. W..

Huber. R.. Roitsch. C. & Fenton. J. W. (1990) The structureof a complex of recombinant hirudin and human -thrombin.Science 249. 2 7 7 - 2 8 0 .8. Grutter. M. G.. Priestle. J. P.. Rahuel. J.. Grossenbacher. H..Bode. W.. H ofsteenge. J. &Stone. S. R. (1990) Crystal struc-ture of thrombin-hirudin complex: a novel mixle of serineprotease inhibition. EMBOJ. 9. 2361-2.^65.9. Tripier. D. (1988) A family of iso-proteins. isolation and se-quence determination of new hirudin. Folia Haematol.lEeipz.) 115. 3 0 - 3 5 .10. Scharf. M.. Engels. J. & Tripier. D. (1989) Primary structure ofnew iso-himdins. EEBS Lett. 225. 10 5- 110.11. Sawyer R. T. (1986) in Uech hiotoay ami tiehaviour. vol. 11. p.420. Oxford University Press. Oxford.

H i n d l l l ol igo

o l i ( o 2O a^

Ball nl iKO .1G T ' n X : T T A C A C C G A C T ( J : 7 \ C C G A A T C I G G 3' i ( X A G A A C T A C T C C C T C T T G C G T T C g r T C T A A C G T T T G C G G T C y A G G T A A A A A C T C C C A G C T G T C T T ^CAViGAATGTGGCTX5ACGTGGCTTAGACCGGTCT 5 3 T G A T G A C G G A C A C X S i a f iC C A A G A T T G C A A A C G C C A C n C C A T T T T r e A aS S T C G Z i C A G A A G Aoligo 4

oligo 5'IX VT.TAACCAGl'CCGTTCAC VAf Aa-A rTGGTCAa3CAAGTa:CAC 5 V TTCCATGGaXn-TTGCCirrAGAGTCTCACTIXXACTGAAGCTIXrrnAAGGCC^^oligo 6

R a m l l lTAi n'AAtJ f

Fig.8. Nucleotide sequence of the six oligonucleotides coding forHM2. The portion coding for the OmpA leader peptide is shown mbold face. The restriction sites HinttUl and Bam[U were introduced to facilitate cloning.


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30 412. Fareed. J.. Walenga. J. M.. Iyer. L.. Hoppensteadt, D. & Pifarre.R. (1991) An objective perspective on recombinant hirudin:a new anticoagulant and antithrombotic agent. Blood Coa^u-tation and Eibrinotvsis 2, 135 147.13. Harvey. R. P.. Degryse. E.. Stefani. L.. Schamber. F.. Cazenave.J. P. Courtney. M.. Tolstoshev. P & Lecocq. J. P (1986) Clon-ing and expression of cDNA coding for the anticoagulant hi-rudin from the bloodsucking leech Hiriido medicinatis, Proc.Natt Acad. Sci. USA S3, 1 0 8 4 - 1 0 8 8 .14. Electricwala. A.. Sawyer. R. T.. Powell Jones. C. & Atkinson.T. (1991) Isolation of thrombin inhibitor from the leech Hiru-dinariu m anitlensis. Btood C oaf^ulation Eibrinotysis 2. 83 89 .15. Lundblad. R. L. (1971) A rapid method for the purification ofbovine thrombin and the inhibition of the purified enzymewith phenylmethylsulphonyl fluoride. Biochemistry 10.2 5 0 1 - 2 5 0 6 .16. Capo rale. C . C arrano. L.. Nitti, G.. Poerio, E.. Pucci. P. & Bu o-nocore. V. (1991) Determination of the primary structure ofan -amylase inhibitor from wheat kernel by Edman degrada-tion and fast atom bombardment mass spectrometry. ProteinSequences and Data Analysis 4, 3 - 8 .17. Houmard. J. & Drapeau. G. R. (1972) Staphylocoeeal protease:a proteolytie enzyme specific for glutamyl bonds. Proc. Natt

Acad. Sci. USA 69. 3506-3509 .18. Lu. H. S.. Klein. M. L. & Lai. P-H. (1988) Narrow-bore high-performance liquid chromatography of phenylthiocarbamoyiamino acids and carboxypeptidase P digestion for protein C-terminal sequence analysis. / Chromatof^r 447, 3 5 1 - 3 6 4 .19. Justisz. M. G.. Martinoli. G. & Tertrin. L. (1962) Micro-dosaged'une activite antithrombique: l'hirudine. Butt. Soc. Ctin.Biot. 44, 4 6 1 - 4 9 6 .20 . Krstenansky. J. L. & Mao. S. J. T. (1987) Antithrombin proper-ties of C-term inus of hirudin using sy nthetic un sulfated N -acetyl-hirudin(45-65) . EEBS Lett. 211, 1 0 - 1 6 .21 . Bradford. M. M. (1976) A rapid and sensitive method for thequantitation of microgram quantities of protein utilizing theprinciple of protein-dye binding. Anal. Biochem. 72, 2 4 8 -254.

22 . Sambrook. J.. Fritsch. E. F. & Maniatis. T. (1989) Moteeuctoning: a laboratory manual. Cold Spring Harbor Labotory Press. Cold Spring Harbor NY.23 . Frohman. M. A.. Dush. M. K. & Martin. G. R. (1988) Raproduction of full-length cDNAs from rare transcripts: amfication using a single gene-specific oligonucleotide primProc. Natl Acad Sci. USA S5, 8 9 9 8 - 9 0 0 2 .24 . Kopin. A. S.. Wheeler. M. B. & Leiter. A. B. (1990) Secrestructure of the precursor and tissue distribution of mRNA. Proc. Natl Acad Sci. USA H7, 2 2 9 9 - 2 3 0 3 .25 . Breathnach. R. & Chambon. P. (1981) Organisation and expsion of eukaryotic split genes coding for proteins. Annu. RBiochem. 50, 3 4 9 - 3 8 3 .26 . Csank. C . Taylor. E M. & Martindale. D. W. (1990) N ucpre-mRNA introns: analysis and comparison of intron quences from Tetrahymena thermophita and other eukaryoNucteic Acid Res. / . 5133 - 5141.27 . De Taxis du Poet. P.. Scacheri. E.. Benatti. L.. Nitti. G.. Valsina. B. & Sarmientos. P (1991) Production oft he HV l varof hirudin by recombinant DNA methodology. Btood Coalation and Eibrinotysis 2. 113120.

28 . Erns t . J . F . (1988) Codon usage and gene express ion . TrBioteclmot. 6. 196-199 .29 . Hemdan. E. S.. Zhao. Y.. Sulkowski. E. & Porath. J. (19Surface topography of histidine residues: a facile probe immobilized metal Ion affinity chromatography. Pro(\ NAcad Sci. USA S6. 1 8 1 1 - 1 8 1 5 .30 . Johnson. P H.. Sze. P. Winant. R.. Payne. P W. & Lazar. J(1989) Biochemistry and genetic engineering of hiruSemin. Thromb. Hemostasis 15, 3 0 2 ^ 3 1 5 .31 . Steiner. V.. Knecht. R.. Borsen. K. O.. Gassmann. E.. StoneR.. Rasehdorf. F.. Sehlaeppi. J. M. & Maschler. R. (19Primary structure and function of novel O-glycosylated hdins from the leech Hirudinaria manitlensis, Bioctiemistry2294-2298.


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Documents

HIrudin AA Sequences, CDNA Cloning, Genomic Organization